Download Inductive heating kills cells that contribute to plaque: a proof

Inductive heating kills cells that
contribute to plaque: a proof-of-concept
Angelo Gaitas and Gwangseong Kim
Kytaro Inc., Miami, FL, USA
Electrical and Computer Engineering Department, Florida International University (FIU),
Miami, FL, USA
ABSTRACT
Submitted 15 January 2015
Accepted 13 April 2015
Published 28 April 2015
Corresponding author
Angelo Gaitas, [email protected]
Academic editor
Li Zuo
Additional Information and
Declarations can be found on
page 9
DOI 10.7717/peerj.929
Copyright
2015 Gaitas and Kim
Distributed under
Creative Commons CC-BY 4.0
OPEN ACCESS
Inducing cell death by heating targeted particles shows promise in cancer treatment.
Here, we aim to demonstrate the feasibility of extending the use of this technique to
treat and remove vascular deposits and thrombosis. We used induction heating of
macrophages, which are key contributors to atherosclerosis and have demonstrated
clear feasibility for heating and destroying these cells using ferromagnetic and
pure iron particles. Specifically, iron particles achieved maximum temperatures
of 51 ± 0.5 ◦ C and spherical particles achieved a maximum temperature of
43.9 ± 0.2 ◦ C (N = 6) after 30 min of inductive heating. Two days of subsequent
observation demonstrated that inductive heating led to a significant reduction in
cell number. Prior to induction heating, cell density was 105,000 ± 20,820 cells/ml
(N = 3). This number was reduced to 6,666 ± 4,410 cells/ml for the spherical
particles and 16,666 ± 9,280 cells/ml for the iron particles 24 h after inductive
heating. Though cell density increased on the second day following inductive
heating, the growth was minimal. Cells grew to 26,667 ± 6,670 cells/ml and
30,000 ± 15,280 cells/ml respectively. Compared to cell cultures with iron and
spherical particles that were not subjected to induction heating, we observed a 97%
reduction in cell count for the spherical particles and a 91% reduction for the iron
particles after the first 24 h. After 48 h we observed a 95% reduction in cell growth for
both spherical and iron particles. Induction heating of microparticles was thus highly
effective in reducing the macrophage population and preventing their growth. These
results demonstrate the feasibility of targeting cells involved in atherosclerosis and
warrant further research into potential clinical applications.
Subjects Bioengineering, Biophysics, Drugs and Devices, Translational Medicine
Keywords Remote cell death, Microparticles, Atherosclerosis treatment methodologies, Electro-
magnetic induction heating, Translational research
INTRODUCTION
Electromagnetic induction heating is the heating of an electrically conducting object (in
this case microparticles) by an alternating magnetic field created by a high-frequency
alternating current (AC) passing through a coil (Semiatin, 1988). Generated Eddy currents
on and within the particles lead to Joule heating (Semiatin, 1988). Induction heating
was first suggested as a means for therapy (Gilchrist et al., 1957) in 1957, and it has been
proposed for cancer therapy to reduce the size of tumors by specifically targeting tumor
How to cite this article Gaitas and Kim (2015), Inductive heating kills cells that contribute to plaque: a proof-of-concept. PeerJ 3:e929;
DOI 10.7717/peerj.929
1 http://www.nhlbi.nih.gov/health/
health-topics/topics/atherosclerosis/
treatment.html
2 http://www.cdc.gov/heartdisease/facts.
htm
cells with nanoparticles, which are then heated to high enough temperatures to cause death
of cancerous cells (Jordan et al., 1997; Jordan et al., 1999; Van der Zee, 2002; CF Chan & B
Kirpotin, 1993). Given the promising results for application of inductive heating in cancer
therapeutics, we have aimed to demonstrate the feasibility for applying this method to
other pathologies. Specifically, we have chosen to test the ability of inductive heating to
destroy cells associated with arthrosclerosis and thrombosis, as prevention strategies and
treatments for these conditions are limited in their effectiveness.
In atherosclerosis, chronic inflammation of the arterial wall is caused by the buildup
of macrophages, white blood cells, platelets, and other particles that form plaque (Maton
et al., 1993). Following plaque formation, stenosis and aneurysm occur, and plaque may
eventually rupture and cause acute coronary events (Lusis, 2000). In thrombosis, a blood
clot is formed inside a blood vessel made from platelets and fibrin (Furie & Furie, 2008).
The clot obstructs blood flow, ultimately creating anoxia and tissue death. A clot may also
break free becoming an embolus.
Current treatment methodologies for atherosclerosis include life style change,
medication, and surgical interventions.1 Life style changes and medications largely rely on
delaying the progress of plaque buildup rather than removing it. The surgical interventions
are limitedly performed for severe atherosclerosis cases (see footnote 1). Treatment for
thrombosis mainly involves the use of anticoagulant medication. While anticoagulant
therapy prevents worsening of thrombosis, it does not remove the thrombus and comes
with various risks, including bleeding, recurrence of thrombosis, pulmonary embolism,
and post-thrombotic syndromes. There is thus a great need for treatements that can
reduce the pre-existing vascular buildups and restore the normal circulation while avoiding
invasive surgical procedures.
Here we have employed a method of inductive heating of micrometer-sized particles to
break up vascular deposits and thrombus, which are associated with a number of health
complications,2 to test if this method can effectively destroy these problematic cells. Our
results show significant reduction in cell number as a result of inductive heating and
support the view that inductive heating as a therapeutic option should be more extensively
studied.
MATERIALS AND METHODS
Materials
In this preliminary work, we used spherical carboxyl ferromagnetic particles, prepared
using chromium dioxide and coated onto uniform polystyrene particles, at a concentration
of 0.5% w/v and diameter 28.0–34.9 µm (catalog # CFM-300-5 from Spherotech). We also
used pure iron particles <44 µm (powder Fe-110 from Atlantic Equipment Engineers).
An induction heating unit was used externally to create an alternating magnetic field
strong enough to heat those particles and reduce cell buildup (as shown conceptually in
Fig. 1A). Micro- and nano- particles have been extensively studied as carrier devices to
deliver drugs or functional materials to target sites and release/actuate the payload in a
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Figure 1 (A) is an illustration of micron-sized particles attached on part of vascular deposit site with
at least one type of a biological binder, such as an antibody. Inductive heating is used to reduce vascular
deposits by heating the targeted vascular deposits at relevant temperatures. (B) The set-up used to heat
cells with the micro-particles. (C) Macrophages in vial. (D) Macrophages in vial with spherical particles
(diameter 28.0–34.9 µm) attached after 2 h of incubation (×200 magnification).
controlled manner (Rabinow, 2004; Reddy et al., 2006; Kumar, 2000; Brannon-Peppas &
Blanchette, 2012; Birnbaum & Brannon-Peppas, 2004; Torchilin, 2011).
Nanoparticles are widely used for targeted delivery approaches based on their ability to
penetrate through the vascular wall to deeper tissue, the ease with which cells uptake them,
and the minimal physical disturbance they cause cells due to their smaller size. However,
microparticles have been used more frequently for controlled release applications based
on biodegradation. It was recently reported that micrometer sized particles can bind
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more efficiently to vascular walls than nanometer sized particles can (Charoenphol et al.,
2011; Charoenphol et al., 2012), making micro-size particles more appropriate for our
application.
Cell culture
We chose macrophages as a model cell line that represents the atherosclerotic condition.
Macrophages are the main component of arterial plaque along with platelets and white
blood cells. Macrophages play a central role in atherosclerosis, including scavenging of
modified lipoprotein, generation of foam cells, breaking down/thinning of the endothelial
layer (fibrous cap) to turn into unstable lesions, secretion of cytotoxic molecules to
promote death of surrounding cells, forming necrotic core, and so on (Moore & Tabas,
2011; Schrijvers et al., 2007; Dickhout, Basseri & Austin, 2008). Reduction of macrophages
can help reduce atherosclerosis (Croons, Martinet & RY De Meyer, 2010; Saha et al., 2009;
Tabas, 2005). A number of recent studies suggested using macrophage as therapeutic target
for artherosclerosis (Glass, 2002; Demidova & Hamblin, 2004; Amirbekian et al., 2007). In
addition, macrophages are typical phagocytotic cells. In the immune system macrophages
ingest pathogenic microorganisms and have the ability to bind randomly to foreign
particles including the micro-particles we introduced. The non-specific phagocytosis
process was utilized to simulate cell binding to microparticles. The microparticles used in
this study were not modified for targeting.
Our inductive heating was performed on a murine macrophage cell line, RAW 264.7,
These macrophages (Fig. 1C) were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) and supplemented with 10% fetal bovine serum and 1% Pen-Strep. For the
induction experiments, cells were seeded in 4 mL glass vials (40,000 cells) instead of in a
culture flask.
Cell death by heating
To determine the temperature required to achieve necrosis of cells, we placed the vials
on a temperature controlled heating plate (Lakeshore temperature controller with 0.1 K
accuracy). The temperature was measured with both a thermocouple (Omega, Stamford,
Connecticut, USA) in contact and an infrared thermometer (Infrared Thermometer Dual
Focus LS; Micro-Epsilon, Raleigh, North Carolina, USA) remotely. The vials were kept in
an incubator overnight to allow surviving cells to grow. The following day, macrophage
cells were lifted by trypsinization at room temperature, and vigorously flushed. The
number of cells in suspension was determined by hemocytometry.
Cell death by induction heating
Macrophage cells were prepared in the 4 mL vials (40,000 cells/mL input). The following
day, microparticles were added into the cell solution at 0.1 mg/mL final concentration and
incubated for a minimum of 2 h to allow for cells to attach (Fig. 1D). The vials were placed
in the center of the induction coil. Induction heating was generated using an Easyheat
induction heating system (Ambrell-Ameritherm Company, Scottsville, New York, USA).
A picture of the set-up is shown in Fig. 1B. An infrared thermometer was placed under
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Figure 2 Temperature change over time of particles on cells in vials and in the alternating magnetic field
with standard error bars (sample number N = 6).
the vial to measure the temperature of the cells and the microparticles. The frequency
used was 350 kHz, the current through the coil was 178.5 A, and the power was 1 kW,
which provided a field intensity of 34 kA/m at the center of the coil. After completing the
heating procedure, the cells were kept in an incubator (37 C, 5% CO2 , and nearly saturated
moisture) overnight. The following day, the cells were detached from the glass bottom by
trypsinization at room temperature and by vigorous flushing with a pipette. The cells were
mixed with the equivalent volume of trypan blue solution to discriminate live cells from
dead ones and counted with a hemocytometer using an Olympus IMT-2 Inverted Phase
Contrast Fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan). The process was
repeated a day later with the remaining vials.
In addition to our test group, we employed two control groups. A batch of cells, which
were targeted with particles that did not undergo inductive heating served as one control,
to ensure that the particles themselves were not cause for cell death through, for example,
toxicity. We also used a batch of cells without targeted particles as a separate control. We
inductively heated 6 vials from each of our test group on day zero. After 24 h, we counted
cells from half of the vials from each group, and following another 24 h, we counted cells
from the remaining vials.
RESULTS
Using our induction heating process (Fig. 1), we first tested what temperature was required
to achieve necrosis of cells. To do this, we placed vials on temperature-controlled heating
plates, and we set the temperature to 37 ◦ C, 45 ◦ C and 55 ◦ C for 30 min each. After 24 h,
we found that there were about 40,000 cells/ml at a temperature of 37 ◦ C, 14,000 cells/ml
heated to 45 ◦ C and no cells survived (0 cells/ml) at temperatures of 55 ◦ C.
Induction heating results of the two particle types on cell culture in media are shown
in Fig. 2. The iron microparticles achieved a maximum temperature of 51 ± 0.5 ◦ C
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after 30 min (N = 6), while the spherical particles achieved a maximum temperature of
43.9 ± 0.2 ◦ C (N = 6). It should be noted that these values represent the temperature of
the entire solution. Heating by induction is spatially confined within the short vicinity of
particles. Therefore, the effective temperature at the particle may be much higher than the
bulk temperature measured by the IR thermometer.
We investigated the differences in cell number across groups as a way to determine the
actual impact of inductive heating on cell death. Significant reduction in cell survival was
observed in inductively heated cells, compared to the controls, as shown in Fig. 3. While
the initial number of cells was 105,000 ± 20,820 cells/ml, 24 h after induction heating
the cell count was reduced to 6,666 ± 4,410 cells/ml for the spherical particles and to
16,666 ± 9,280 cells/ml for the iron particles (N = 3). After an additional 24 h, the cell
count was 26,667 ± 6,670 cells/ml for the spherical particles and 30,000 ± 15,280 cells/ml
for the iron particles respectively (N = 3). In contrast, the cells that with no particles
grew to 226,670 ± 23,330 cells/ml after 24 h and to 921,670 ± 78,550 cells/ml after
48 h. Further, the cells with spherical particles that were not subjected to heating grew
to 216,660 ± 42,262 cells/ml after 24 h and 516,660 ± 76,720 cells/ml after 48 h. Cells
with iron particles that were not heated grew to 188,333 ± 4,409 cells/ml after 24 h and to
648,333 ± 151,116 cells/ml after 48 h. (P < 0.002 of controls vs. ferromag. day 1 and iron
day 1, t-test. P < 0.001 of controls vs. ferromag. day 2 and iron day 2, t-test.)
DISCUSSION
The present study demonstrates that induction heating of two types of microparticles
can feasibly cause effective damage to macrophage cells, which is a cell line that is highly
relevant to atherosclerosis. Though induction heating has been primarily investigated in
the context of cancer therapy, we believe that its application to cancer should be extended
and that we should begin investigating the potential to use induction heating to treat
vascular buildups. These results can help inform therapeutic parameters as translational
research is pursued, including the effective particle size, type of particles, and possible
temperature range that could be effective for destroying macrophages in atherosclerosis.
Our findings demonstrate that spherical particles have a better ability to bind to cells
than random shaped iron particles. While most of the spherical magnetic particles attached
to cellular surfaces, iron particles were only partially uptaken by macrophage cells. The
highest temperature achieved by spherical magnetic particles was ∼43 ◦ C, but cell death at
this temperature was greater than that achieved by the iron particles, which were heated at
higher temperatures (∼51 ◦ C).
Our study included some limitations. For instance, we observed that cells without
microparticles exhibited higher growth compared to the controls that included microparticles but were not subjected to heating. This reduction in cell growth may be due to the
activation of the phagocytotic activity of the cells rather than the toxicity of the particles,
because the exponential growth pattern was still observed.
The heating of metal particles by induction was confined to the narrow vicinity around
the particle. The temperature in the local region could therefore be much higher than bulk
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Figure 3 (A) Heated spherical particles on cells on day 2 (mag. ×200). (B) Heated iron particles on
cells on day 2 (mag. ×200). (C) Control vial on day 2 (mag. ×200). (D) Spherical particles on cells in
control vials on day 2 (mag. ×200). (E) Iron particles on cells in control vials on day 2 (mag. ×200).
(F) Hemocytometry cell count of cell growth. Results with standard error of mean (N = 3).
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temperature we measured. The tighter contact of cells with the spherical microparticles
may explain the results. Ultimately, we believe that the local temperature should be high
enough to cause cell lysis, not just necrosis because these temperatures would ensure
reduction of the plaque or thrombus, while avoiding creating an embolus.
Future research should address the several challenges related to treating plaque build-up
in atherosclerosis and thrombosis with heat induction. For example, heating can cause
platelet aggregation, leading to an inflammatory response or clotting. This issue could
potentially be addressed by fabricating microparticles whose half surface that does not stick
to organic materials such as platelets, while the other half is coated with target antibodies.
Determining the ideal number and size of microparticles to use during heat induction
should also be investigated in future studies.
Eventually, surface functionalization of particles will be necessary to selectively deliver
the particles to arterial plaque. A biological targeting moiety (antibodies, peptides, aptamers etc.) attached to the particles that will selectively bind to one of the substances that
makes up vascular deposits would be required when flow is introduced. The methodology
for specific targeting to arterial plaque is not well established yet. The microparticles can,
for instance, be coated with more than one binder, mimicking monocytes which initiate
plaque buildup. In one example, one antibody may aid in rolling adhesion (selectin) and
another antibody can be used for stationary adhesion (integrin).
An advantage of induction heating is that the therapeutic efficacy can be activated only
where the magnetic field is applied. This localization could minimize the possible side
effects of non-specific uptake of particles by other distant organs. Thus the accumulation
of particles elsewhere may not be damaging because they will not be heated.
In this introductory work we used commercially available particles. However, microparticles can also be manufactured with microfabrication methods such as surface micromachining to control their size, dimensions, properties and function. The magnetic particles
can also be used in combination with (or as part of) a magnetic resonance imaging (MRI)
contrast agents for monitoring the vascular deposits with MRI contrast enhancement while
the patient is undergoing treatment (Ruehm et al., 2001; Yu et al., 2000).
In a real life setting microparticle extraction would be necessary. The particles can be
extracted from a patient by placing a magnet in proximity to the area of extraction causing
the particles to flow out of a blood vessel. Alternatively, the microparticles can be fabricated
with biodegradable materials containing magnetic payloads to facilitate clearance from the
body. Thus, there are a number of options and opportunities related to the development
of techniques using heating induction with microparticles to treat atherosclerosis and
thrombosis specifically, as well as other diseases.
CONCLUSIONS
In this manuscript, we have demonstrated the feasibility of destroying macrophages, which
contribute to the plaque associated with arthrosclerosis and thrombosis, using inductively
heated micrometer-sized particles. The results indicate that the technique was highly effective in reducing a cell culture population. We have attempted to introduce the concept and
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possibility of using induction heating in conjunction with microparticles for therapeutic
applications in medicine. Though much research is required before application of this
technique could be clinically implemented, our study demonstrates the potential value of
the technique and suggests that further research in this area could provide clinical benefits.
ACKNOWLEDGEMENTS
The authors thank Ambrell-Ameritherm Co. for loaning us the induction heating system.
We would also like to thank Dr. Flicker for her support.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This research was funded by National Institutes of Health (grant # GM084520). The
funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
National Institutes of Health: GM084520.
Competing Interests
The authors are employees of Kytaro, Inc.
Author Contributions
• Angelo Gaitas and Gwangseong Kim conceived and designed the experiments,
performed the experiments, analyzed the data, contributed reagents/materials/analysis
tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/
10.7717/peerj.929#supplemental-information.
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